Field
This invention generally relates to a composition and method of using recombinant small RNAs, such as small interfering RNAs (siRNA), microRNAs (miRNA) and their hairpin-like precursors (pre-miRNA), as tumor suppressing anti-cancer drugs for treating human tumors and cancers, in particular, but not limited, for treating skin (melanoma), blood (leukemia), prostate, breast, liver and lung cancers as well as various neoplastic tumors, such as brain tumors and teratocarcinomas that contain a variety of tumorous and cancerous cells derived from all three germ layers of tissues, including ectoderm, mesoderm and endoderm. More specifically, the present invention relates to the use of man-made miR-302-like siRNA (siR-302) and/or miRNA precursors (pre-miR-302) for developing novel medicines for a variety of anti-cancer therapies, in particular for human lung cancer treatments. These siRNA/pre-miR-302 drugs can be produced in prokaryotes as expression-competent DNA vectors and/or the resulting hairpin-like RNA products. As prokaryotic cells do not naturally transcribe or process hairpin-like RNAs, of which the structure resembles a transcriptional termination code in the gene expression systems of prokaryotes, and further in view of the lack of several essential enzymes such as type-II RNA polymerases (Pol-2) and RNaseIII Dicers in prokaryotes, the present invention further teaches a novel gene expression method for expressing pre-miRNAs in prokaryotes, or called prokaryote-produced miRNA precursors (pro-miRNA), using a newly found hairpin-like RNA transcription mechanism in prokaryotes (which is first disclosed in our priority of U.S. patent application Ser. No. 13/572,263). In addition, since miR-302 is a tumor-suppressor miRNA in human embryonic stem cells (hESCs), our findings presented in this invention can be further used to design and develop new medicines, vaccines and/or therapies useful for treating other tumor- and cancer-associated diseases.
Description of the Related Art
Stem cells are a resourceful treasure box containing numerous effective ingredients useful for stimulating new cell growth and tissue regeneration, repairing or rejuvenating damaged/aged tissues, treating aging-related diseases, and preventing tumor formation and cancer progression. Hence, it is conceivable that we can use these stem cells as a tool for screening, identifying and producing these stem cell-specific ingredients for developing novel medicines and therapies directed against a variety of human diseases. As a result, the medicines and therapies so obtained can be used in many pharmaceutical and therapeutic applications, such as a biomedical kit, device and apparatus for research, diagnosis, and/or treatment, or a combination thereof.
MicroRNA (miRNA) is one of the main effective ingredients in human embryonic stem cells (hESCs). Major hESC-specific miRNA species include, but not limited, members of the miR-302 family, miR-371˜373 family, and miR-520 family. Among them, the miR-302 family has been found to play a functional role in tumor suppression (Lin et al., 2008 and 2010; U.S. Pat. Nos. 9,394,538, 9,399,773, and 9,422,559 to Lin et al.). MiR-302 contains eight (8) familial members (miR-302s), including four (4) sense miR-302 (a, b, c, and d) and four (4) antisense miR-302* (a*, b*, c*, and d*). These sense and antisense miRNA members are partially matched and can form double-stranded duplexes to each other. Precursors of miR-302 are formed by miR-302a and a* (pre-miR-302a), miR-302b and b* (pre-miR-302b), miR-302c and c* (pre-miR-302c), and miR-302d and d* (pre-miR-302d) with a link sequence in one end (stem loop). In order to activate miR-302 function, miR-302 precursors (pre-miR-302s) are first processed into mature miR-302s by cellular RNaseIII Dicers and further form RNA-induced silencing complexes (RISCs) with certain argonaute (AGO) proteins, subsequently leading to either RNA interference (RNAi)-mediated direct degradation or translational suppression of many targeted gene transcripts (mRNAs), in particular including some major oncogene mRNAs as disclosed in our previous U.S. Pat. Nos. 9,394,538, 9,399,773, and 9,422,559 (Lin et al).
MiR-302 is the most abundant non-coding (ncRNA) species found in hESCs and induced pluripotent stem cells (iPSCs). Our previous studies have shown that ectopic overexpression of miR-302 over the level found in hESC H1 or H9 cells is able to reprogram both human normal and cancerous cells to hESC-like iPSCs with almost no tumorigenecity similar to the pluripotent stem cells of a morula-stage early human zygote (Lin et al., 2008, 2010 and 2011; FP 2198025 to Lin et al.; U.S. patent application Ser. No. 12/149,725 and Ser. No. 12/318,806 to Lin et al.; U.S. Pat. No. 9,394,538 to Lin et at). Relative quiescence is a well defined character of these miR-302-induced iPSCs, whereas late blastocyst-derived hESCs and other previously reported three-/four-factor-induced (either Oct4-Sox2-Klf4-c-Myc or Oct4-Sox2-Nanog-Lin28) iPSCs all present a very fast cell proliferation rate (12-15 hours/cycle) similar to that of a neoplastic tumor/cancer cell (Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007; Wang et al., 2008). To disclose this tumor suppression effect of miR-302, we are the first researchers to identify the involvement of two miR-302-targeted G1-checkpoint regulators, including cyclin-dependent kinase 2 (CDK2) (Lin et al., 2010; U.S. Pat. No. 9,394,538 to Lin et al.) and BMI-1 (Lin et al., 2010; U.S. Pat. No. 9,422,559 to Lin et al). Our studies found that miR-302 concurrently silences these two major target genes to inhibit both of the cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways during the G1-S transition of cell cycle, so as to prevent the tumorigenecity of pluripotent stem cells. Furthermore, we also found that the reprogramming function of miR-302 can reprogram high-grade malignant cancers into a low-grade benign or even almost normal state (U.S. Pat. No. 9,399,773 to Lin et al.), via a partial reprogramming mechanism.
However, it is not known whether these previously found tumor suppression functions of miR-302 can be directly used for human lung cancer therapy. Our previous patents and their associated applications can not determine this possibility in view of the varieties of lung cancer types. Unlike other human cancers, the pathological causes of lung cancers are complicated, including air pollution, smoking, asbestosis, virus, long-term inflammation, genetic mutation, and metastasis of other cancers into lung tissues, or even a combination there of. Due to such a great complexity, even an expert can not easily predict the success of a new therapy in other cancers for treating lung cancers. There may require the involvement of more novel therapeutic mechanisms to deal with the complexity of lung cancers.
There is no direct genetic link between miR-302 and lung cancer. The genomic sequence encoding miR-302 is located in the 4q25 locus of human chromosome 4, a conserved region frequently associated with longevity. More precisely, miR-302 is encoded in the intron region of the La ribonucleoprotein domain family member 7 (LARP7) gene and expressed via an intronic miRNA biogenesis pathway that we found (Ying and Lin, 2004; Barroso-delJesus, 2008; FIG. 13; SEQ.ID.NO.5). In our previous studies, we had observed that introduction of miR-302 can stimulate the expression of many other hESC-specific miRNAs, such as miR-92, miR-93, miR-367, miR-371˜373, miR-374, and the miR-520 familial members in the transfected cells (Lin et al., 2008, 2010 and 2011; FP 2198025 to Lin et al.; U.S. patent application Ser. No. 12/149,725 and Ser. No. 12/318,806 to Lin et al.). Analyses using the online “TARGETSCAN” and “PICTAR-VERT” programs further revealed that miR-302 shares over 400 target genes with these stimulated miRNAs, suggesting that they may also play a similar functional role as mir-302. These shared target genes include, but not limited, members of RAB/RAS-related oncogenes, EC T-related oncogenes, pleiomorphic adenoma genes, E2F transcription factors, cyclin D binding Myb-like transcription factors, HMG-box transcription factors, Sp3 transcription factors, transcription factor CP2-like proteins, NFkB activating protein genes, cyclin-dependent kinases (CDKs), MAPK/JNK-related kinases, SNF-related kinases, myosin light chain kinases, TNF-alpha-induce protein genes, DAZ-associated protein genes, LIM-associated homeobox genes, DEAD/H box protein genes, forkhead box protein genes, BMP regulators, Rho/Rac guanine nucleotide exchange factors, IGF receptors (IGFR), endothelin receptors, left-right determination factors (Lefty), cyclins, p53 inducible nuclear protein genes, RB-like 1, RB binding protein genes, Max-binding protein genes, c-MIR cellular modulator of immune recognition, Bcl2-like apoptosis facilitator, protocadherins, TGFβ receptors, integrin 134/138, inhibin, ankyrins, SENP1, NUFIP2, FGF9/19, SMAD2, CXCR4, EIF2C, PCAF, MECP2, histone acetyltransferase MYST3, nuclear RNP H3, and many nuclear receptors and factors. The majority of these target genes are involved in embryonic development and tumorigenecity. Hence, it is conceivable that miR-302 can further stimulate its homologous miRNAs, such as miR-92, miR-93, miR-367, miR-371˜373, miR-374, and miR-520, to enhance and/or maintain its functionality.
Although miR-302 is useful for designing and developing novel anti-cancer drugs/vaccines, its production is problematic because natural miR-302 can only be found in human pluripotent stem cells such as hESCs, of which the original source is very limited and highly controversial. Alternatively, synthetic small interfering RNAs (siRNA) may be used to mimic pre-miR-302; yet, since the structure of a pre-miR-302 is formed by two mis-matched strands of miR-302 and miR-302*, those perfectly matched siRNA mimics can not replace the function of miR-302*, of which the sequence is totally different from the antisense strand of siRNA. For example, the antisense strand of siRNA-302a mimic is 5′-UCACCAAAAC AUGGAAGCAC UUA-3′ (SEQ.ID.NO.1), whereas native miR-302a* is 5′-ACUUAAACGU GGAUGUACUU GCU-3′ (SEQ.ID.NO.2). As the full miR-302 function must result from both of its sense miR-302 and antisense miR-302* strands, many previous reports using siRNA mimics have shown different results from native miR-302 functions in nature. On the other hand, our recent discovery of iPSCs may provide an alternative solution for pre-miR-302 production (EP 2198025 to Lin et al.; U.S. patent application Ser. No. 12/149,725 and Ser. No. 12/318,806 to Lin et al.). Nevertheless, the cost and risk of growing these iPSCs is still too high to be used for industrial production now.
Alternatively, the use of prokaryotic competent cells may be a possible approach for producing human miRNAs and their precursors (pre-miRNAs). However, prokaryotic cells lack several essential enzymes required for eukaryotic miRNA expression and processing, such as Drosha and Dicer proteins. Also, prokaryotic RNA polymerases do not efficiently transcribe small RNAs with high secondary structures, such as hairpin-like pre-miRNAs and shRNAs. In fact, there is no true miRNA species encoded in bacterial genomes and bacteria do not naturally express miRNA because hairpin-like RNA structures like pre-miRNAs are similar to the intrinsic transcription termination codes in prokaryotic gene expression systems (McDowell et al., Science 1994). As a result, if we can express human miRNAs in prokaryotes, the resulting miRNAs will remain in their precursor forms similar to pri-miRNA (a large primary cluster of multiple pre-miRNAs) or pre-miRNA (one single hairpin RNA). Yet, as aforementioned, the real challenge is how to force the expression of human miRNAs in prokaryotes. To overcome this problem, our priority inventions of U.S. patent application Ser. No. 13/572,263, Ser. No. 14/502,608 and Ser. No. 14/527,439 have established a method for generating prokaryote-produced microRNAs (pro-miRNA). The pro-miRNAs so obtained have been tested to possess the same sequences, structures and functions as their native pre-miRNA counterparts.
As learning from current textbooks, every ordinary skill person in the art knows very well that prokaryotic and eukaryotic transcription machineries are very different and hence not compatible to each other. For example, based on current understandings, eukaryotic RNA polymerases do not bind directly to a promoter sequence and require additional accessory proteins (cofactors) to initiate transcription, whereas prokaryotic RNA polymerases are single holoenzymes that bind directly to a promoter sequence to start transcription. It is also a common knowledge that eukaryotic messenger RNA (mRNA) is synthesized in the nucleus by type-II RNA polymerases (Pol-2) and then processed and exported to the cytoplasm for protein synthesis, while prokaryotic RNA transcription and protein translation take place simultaneously off the same piece of DNA in the same place. This is because prokaryotes such as bacteria and archaea do not have any nucleus-like structure. Accordingly, these natural differences make a prokaryotic cell difficult or even impossible to produce eukaryotic RNAs using eukaryotic promoters.
Prior arts attempt at producing mammalian peptides and/or proteins in bacterial cells, such as U.S. Pat. No. 7,959,926 to Buechler and U.S. Pat. No. 7,968,311 to Mehta, used bacterial or bacteriophage promoters. For initiating expression, a desired gene was cloned into a plasmid vector driven by a bacterial or bacteriophage promoter. The gene must not contain any non-coding intron because bacteria do not have any RNA splicing machinery to process the intron. Then, the vector so obtained was introduced into a competent strain of bacterial cells, such as Escherichia coli (E. coli), for expressing the transcripts (mRNAs) of the gene and subsequently translating the mRNAs into proteins. Nevertheless, the bacterial and bacteriophage promoters, such as Tac, Lac, Tc, T1, T3, T7, and SP6 RNA promoters, are not Pol-2 promoters and their transcription activities tend to be an error-prone process, which causes mutations and can not express any hairpin-like RNA structure as reported by McDowell et al (Science 1994). In addition, Mehta further taught that glycerol/glycerin might be used to increase the efficiency of bacterial transformation; yet, no teaching was related to enhancement of RNA transcription, in particular Pol-2 promoter-driven prokaryotic RNA transcription. Due to lack of compatibility between eukaryotic and prokaryotic transcription systems, these prior arts were still limited by the use of prokaryotic RNA promoters for gene expression in prokaryotes and none of them were useful for expressing hairpin-like RNAs, such as pre-miRNAs and shRNAs.
Due to system incompatibility, there was no means for producing pre-miRNA/shRNA-like drugs in prokaryotes before our invention of pro-miRNAs. Also, a pre-miRNA/shRNA is sized about 70˜85-nucleotides in length which is too large and costly to be made by a RNA synthesis machine. To overcome these problems, the present invention adopts pro-miRNAs. By adding some defined chemical inducers mimicking eukaryotic transcriptional cofactors, we can create a novel adaptation environment for prokaryotic cells to use eukaryotic Pol-2 and Pol-2-like viral promoters for transcribing hairpin-like pre-miRNAs and shRNAs. The advantages are: first, cost-effective mass production due to the fast growth of bacteria; second, easy handling because of no need for growing dedicate hESCs or iPSCs; third, high fidelity productivity in terms of Pol-2 promoter-driven RNA transcription; fourth, high purity of resulting pre-miRNAs and siRNAs due to lack of true miRNA in prokaryotes; and last, no endotoxin, which can be further removed by certain chemical treatments. Therefore, a method of producing high quality and high quantity of pro-miRNAs as drugs for treating human lung cancers is highly desirable.